JP2013134978A - Sample support for transmission electron microscope, transmission electron microscope and three-dimensional structure observation method of sample - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a versatile sample support capable of acquiring the 360° omnidirectional information simply and accurately in three-dimensional structure observation using a transmission electron microscope, and to provide a three-dimensional structure observation method.SOLUTION: The sample support for a transmission electron microscope includes a sample table provided, at the tip thereof, with a crystalline sample pedestal at least a part of which is single crystal, and a support substrate to which the sample pedestal is fitted removably. The sample pedestal can be fitted to the support substrate removably, even if it is rotated about the axis of rotating. The transmission electron microscope includes a sample holder on which a sample is mounted, and a sample movement mechanism which adjusts the position and the inclination angle of the sample holder. The transmission electron microscope includes the sample support on the sample holder. There is also provided a three-dimensional structure observation method of sample using the transmission electron microscope.

Description

  The present invention relates to a sample support for a transmission electron microscope, a transmission electron microscope, and a method for observing a three-dimensional structure of a sample. Especially when acquiring continuous sample tilt images for three-dimensional structure observation using a transmission electron microscope, it is possible to easily acquire a high tilt sample tilt image that becomes an information-deficient area and to accurately specify the sample tilt angle. It relates to a sample support that can be used.

  In recent years, there has been an increasing need to observe a three-dimensional structure of a minute sample using a transmission electron microscope or a scanning transmission electron microscope. However, basically, a transmission electron microscope can only obtain a two-dimensional projection image of a sample. Therefore, in order to observe the three-dimensional structure, first, the sample is continuously tilted in the apparatus, and projection images from many directions are taken. Thereafter, a method of reconstructing a three-dimensional image by processing the projection image group with a computer is employed. This series of observation techniques is called electron beam tomography observation. In this method, in order to accurately reconstruct a three-dimensional image from a group of two-dimensional projection images, the projection images from all directions are photographed at as fine an angular interval as possible, and the positions and orientations between the respective projection images. Accurate alignment is required.

  Furthermore, in order to take the above-mentioned continuous sample tilt image, the thinned and thin pillars are formed at the end of the sample stage where a notch mesh (a half-moon shaped metal plate) or a grid-like metal mesh is processed. The observed sample was fixed. This was mounted on a standard sample holder for a transmission electron microscope, and projected images from many directions were taken using the sample tilt mechanism of the electron microscope. However, in a general electron microscope, the sample tilt angle is limited due to restrictions on the apparatus (generally ± 60 ° to ± 70 ° is the upper limit), and a sample tilt angle region (information missing region) that cannot be observed is generated. This information missing region is known to cause distortion in the reconstructed three-dimensional image and to anisotropically reduce the spatial resolution.

  On the other hand, in Patent Document 1, a special sample holder provided with a mechanism capable of rotating the sample stage portion, which fixes the sample at the tip of the sample holder, by 360 ° in the electron microscope is used. There has been proposed a sample stage in which the direction of the sample stage can be confirmed by forming a mark. This makes it possible to observe the sample from all directions while confirming the direction during processing and observation of the sample.

  Further, in Patent Document 2, a sample support provided with a thin plate-shaped base, a sample support member for fixing a sample that can be freely rotated and fixed with respect to the base, and a protractor showing a rotation angle so as to surround the sample support member. A stand has been proposed. This is mounted on a standard transmission electron microscope sample holder, and by combining the rotation angle of the sample support member and the tilt angle of the sample stage tilt mechanism of the electron microscope, the sample can be observed from all directions.

JP 2007-018944 A JP 2009-070604 A

  In Patent Document 1, since a mechanism for rotating a pillar-shaped sample stage by 360 ° is provided, observation without an information missing region is possible. However, a special sample holder for that purpose must be used, and the apparatus that can be used is also limited, so it is difficult to say that it is a versatile observation method.

  On the other hand, in Patent Document 2, versatility is enhanced because there is no information missing region and the sample support base has a shape that can be mounted on a standard sample holder. However, since the structure is such that the rotation angle of the sample support member is read by the scale of the protractor, there is a problem in terms of reading accuracy and reproducibility.

  The present invention has been made in view of such background art, and in a three-dimensional structure observation using a transmission electron microscope, a sample support capable of observing a sample from all directions of 360 °, and a transmission using the same An electron microscope and a three-dimensional structure observation method for a sample are provided.

  A sample support for a transmission electron microscope that solves the above problems includes a sample stage having a crystalline sample pedestal at least a part of which is a single crystal at a tip portion, and a support on which the sample stage is detachably fitted. A sample support including a substrate, wherein the sample stage is detachable from the support substrate even when the sample stage rotates about a rotation axis.

  A transmission electron microscope that solves the above problems is a transmission electron microscope that includes a sample holder on which a sample is mounted, and a sample moving mechanism that adjusts the position and inclination angle of the sample holder. A sample support is provided.

  A method for observing a three-dimensional structure of a sample that solves the above problem is a method of observing the three-dimensional structure of a sample using a transmission electron microscope having the sample holder and a sample moving mechanism. The sample is mounted on the sample support provided in the holder, and the first sample orientation is determined based on one crystal orientation of the crystalline sample pedestal of the sample support. Obtaining a continuous tilt image group, rotating the sample stage of the sample support, determining the second sample direction based on another crystal direction of the crystalline sample base, and then using the sample moving mechanism A step of acquiring a second continuous tilt image group, specifying a sample orientation of the continuous tilt image by estimating the orientation of each continuous tilt image group from the first sample orientation and the second sample orientation; Having a step of reconstructing a three-dimensional image And features.

  The present invention provides a sample support capable of observing a sample from all 360 ° directions in a three-dimensional structure observation using a transmission electron microscope, a transmission electron microscope using the sample support, and a three-dimensional structure observation method of the sample. Can do.

It is a block diagram which shows one embodiment of the sample support body of this invention. It is a block diagram which shows one embodiment of the transmission electron microscope of this invention. It is the schematic which shows the crystal-axis direction of the crystalline sample base in this invention. It is explanatory drawing which shows the rotation procedure of the sample stand at the time of three-dimensional structure observation.

  A sample support for a transmission electron microscope according to the present invention includes a sample stage provided with a crystalline sample pedestal at least a part of which is a single crystal at a tip portion, and a support base on which the sample stage is detachably fitted. The sample support is characterized in that it can be attached to and detached from the support base even if the sample stage rotates about the rotation axis.

  The sample support of the present invention is a sample support for a transmission electron microscope having a mechanism in which a sample stage, which is a part of the sample support, can be attached or detached. The sample stage provided with such a mechanism can observe the sample from various directions by fixing the sample stage at a plurality of rotation angles. In particular, by mounting on a uniaxial tilt holder or a biaxial tilt holder and combining the sample stage rotation mechanism with the sample tilt in the sample moving mechanism of the electron microscope main body, it is possible to observe the sample from all 360 ° directions. In addition, if the tip of the sample stage is equipped with a crystalline sample pedestal that is partially or entirely single crystal, the direction in which the sample stage is facing, based on the electron diffraction pattern of the single crystal part by a transmission electron microscope Can be determined accurately and with good reproducibility.

  In addition, one crystal axis of this single crystal portion is substantially coincident with the rotation axis direction of the sample stage, and another two crystal axes are orthogonal to the rotation axis direction of the sample stage. And the change in the incident direction of the electron beam due to the attachment / detachment / rotation of the sample stage is also limited to a plane orthogonal to the rotation axis direction of the sample stage. Therefore, the change of the electron diffraction pattern can be easily predicted, and the direction in which the sample stage is facing can be determined more easily.

  A transmission electron microscope according to the present invention is a transmission electron microscope including a sample holder on which a sample is mounted and a sample moving mechanism that adjusts a position and an inclination angle of the sample holder, wherein the sample holder supports the sample. It is characterized by having a body.

  According to another aspect of the present invention, there is provided a method for observing a three-dimensional structure of a sample using a transmission electron microscope having the sample holder and the sample moving mechanism. The sample is mounted on the sample support provided in the holder, and the first sample orientation is determined based on one crystal orientation of the crystalline sample pedestal of the sample support. Obtaining a continuous tilt image group, rotating the sample stage of the sample support, determining the second sample direction based on another crystal direction of the crystalline sample base, and then using the sample moving mechanism And acquiring the second continuous tilt image group, accurately identifying the sample orientation of the continuous tilt image by estimating the orientation of each continuous tilt image group from the first sample orientation and the second sample orientation. And reconstruct a 3D image based on that information It characterized by having a that process.

  In the present invention, the change in the sample orientation due to the attachment / detachment / rotation can be corrected by performing the sample orientation determination procedure by the electron diffraction pattern as described above before and after the attachment / detachment / rotation of the sample stage. Thereby, each continuous tilt image before and after attachment / detachment / rotation is treated as one continuous tilt image data, and a three-dimensional structure image can be reconstructed. As a main procedure, first, a sample is fixed to a crystalline sample pedestal, and the sample pedestal is attached to a support base to obtain a sample support. Mount the sample support on the sample holder of an electron microscope that can tilt the sample more than one axis, determine the first sample orientation based on one crystal orientation of the crystalline sample base, and then use the sample moving mechanism One continuous tilt image group is acquired. Next, the sample stage is rotated, and after determining the second sample orientation based on another crystal orientation of the crystalline sample base, a second continuous tilt image group is acquired using the sample moving mechanism. Next, the sample orientation of the continuous tilt image is accurately specified by estimating the orientation of each continuous tilt image group from the first sample orientation and the second sample orientation. Finally, it is a procedure for reconstructing a three-dimensional structure image based on the information.

  Embodiments of the present invention will be described below with reference to the drawings. However, the dimensions, materials, shapes, relative arrangements, and the like described in this embodiment are not intended to limit the scope of the present invention only to those unless otherwise specified.

  A transmission electron microscope according to the present invention is a transmission electron microscope including a sample holder on which a sample is mounted and a sample moving mechanism that adjusts a position and an inclination angle of the sample holder, wherein the sample holder supports the sample. It is characterized by having a body.

  FIG. 2A shows a general configuration diagram of a transmission electron microscope used for the sample support and the three-dimensional structure observation method of the present invention. FIG. 2B is a schematic view of the tip of the sample holder. The transmission electron microscope includes an electron gun 12, a converging lens 13, an objective lens 14, a projection lens 15, a fluorescent plate 16, and a recording device 17. A sample holder 19 carrying the sample 8 can be inserted between the converging lens 13 and the objective lens 14. The electron beam 20 emitted from the electron gun 12 is converged in a dot shape by the converging lens 13 and is irradiated to the sample mounted on the tip of the sample holder 19. The electron beam transmitted and scattered through the sample is imaged by the objective lens 14, further magnified by the projection lens 15, and projected onto the fluorescent plate 16. Further, when recording or the like is necessary, the fluorescent plate 16 can be removed from the electron beam optical path, and the electron beam 20 can be projected onto the recording device 17 installed further below to take an image.

  The sample holder 19 can be adjusted in position, tilt angle and the like by the sample moving mechanism 18. As a result, the mounted sample can be observed from an arbitrary place and from various angles. However, the insertion space for the sample holder 19 is generally very narrow. In particular, regarding the sample tilt, the sample tilt angle is limited in order to avoid collision between the sample holder 19 and the lens. FIG. 2B shows a general overview of the tip portion of the sample holder 19. A general sample holder is a cylindrical rod for passing an electron beam through a portion corresponding to a sample support mounting portion 21 and an electron beam optical path 22 for mounting and fixing a sample support 1 having a sample fixed at the tip. Space 25 is provided. There are various shapes of mechanisms for holding the sample support 1 on the sample support mounting portion 21. The space 25 generally has a disk with a diameter of 3 mm and a thickness of several tens to several hundreds of μm. The shape can be held. Some sample holders have various functions. In general, a sample holder that can observe a sample tilted in a uniaxial or biaxial direction is frequently used.

  Although the scanning transmission electron microscope is not particularly described here, the transmission electron microscope has many similarities in the apparatus configuration, the purpose of use, and the observation method, and the present invention also exhibits the same effect. Hereinafter, it will be called a transmission electron microscope including a scanning transmission electron microscope.

  FIG. 1A shows a configuration diagram of the sample support 1 of the present invention. Further, FIG. 1B shows an overview when the sample is actually mounted and used. The sample support 1 of the present invention comprises a sample stage 3 provided with a crystalline sample base 5 at least a part of which is a single crystal at a tip portion, and a support base 2 on which the sample stage 3 is detachably fitted. The sample support 3 is characterized in that the sample stage 3 can be attached to and detached from the support base 2 even if it rotates about a rotation axis. The sample support 1 is composed of a support base 2 and a sample base 3, and the sample base 3 is composed of a detachable support 4 and a crystalline sample pedestal 5 provided at the tip portion thereof. In FIG. 1B, a sample 8 is fixed to the tip of the crystalline sample base 5. The position where the sample 8 of the crystalline sample base 5 is provided is not particularly limited to the tip, and may be another position.

  The support base 2 has a substantially meniscus shape as if a part of a disk is cut off, and a groove 6 into which the sample table 3 can be inserted is cut in the center of the chord surface 7. In order for the sample support 1 to be mounted on a general transmission electron microscope sample holder 19, it is desirable that the support base 2 is a meniscus member having a diameter of 3 mm and a thickness of about 300 μm.

  The sample table 3 is detachably fitted in the groove 6 provided in the support base 2, and the cross-sectional shape 23 of the sample base and the cross-sectional shape 24 of the groove of the support base are preferably the same. In FIG. 1B, both the cross-sectional shapes 23 and 24 are square. The cross-sectional shape of the sample stage and the cross-sectional shape of the groove of the support base are preferably regular polygons or circles. The cross-sectional shape 24 intersecting the chord surface 7 of the groove 6 may be a polygon or an ellipse such as a quadrangle or a hexagon, but in order for the sample table 3 to be freely rotatable and removable, it may be a square or a regular hexagon. A regular polygon or a circle is desirable. In the case of a circular shape, there is a degree of freedom to rotate at an arbitrary rotation angle, and in the case of a regular polygon, the rotation angle is limited. However, as will be described later, in the procedure of determining the sample orientation from the electron diffraction pattern before and after the attachment / detachment / rotation, it becomes a merit that the change of the electron diffraction pattern due to the attachment / detachment / rotation can be easily predicted, so the regular polygon is also a desirable shape. . The material of the support base 2 may be any material that is non-magnetic, has sufficient mechanical strength, and can be easily processed. Examples thereof include copper, molybdenum, gold, platinum, SUS, and silicon.

  The removable support 4 is a columnar member, and the cross-sectional shape is the same as the cross-sectional shape of the groove so that it can be inserted into and fixed to the groove 6 cut in the support base 2. When inserted into the support base 2, the length should be such that the end of the detachable support 4 coincides with the chord surface 7 or slightly protrudes. This is because when the end of the detachable support 4 is recessed from the chord surface 7, the sample may be hidden behind the support base 2 on the electron beam passage path when the sample is tilted and may not be observed. Because there is. The material may be the same as that of the support substrate, but need not be the same material as the support substrate.

  The crystalline sample base 5 is preferably made of a single crystal in order to determine the sample orientation from the electron diffraction pattern of this portion. Of course, there is no problem as long as the orientation of the sample can be determined and the electron diffraction image is clear to some extent, and any member may be used as long as one single crystal grain occupies a large volume ratio. Hereinafter, the single crystal portion that mainly contributes to the electron diffraction pattern is simply referred to as a single crystal portion.

  The crystal axis of the single crystal portion of the crystalline sample base 5 takes various directions as the sample stage 3 is attached and detached and rotated. When aligning the sample orientation based on the electron diffraction pattern, one crystal axis of the single crystal portion substantially coincides with the rotation axis direction of the sample stage 3, and the other two crystal axes are the rotation axis direction of the sample stage. It is more convenient to be orthogonal. The change in the incident direction of the electron beam due to the attachment / detachment / rotation of the sample stage 3 is limited to a plane orthogonal to the direction of the rotation axis 9 of the sample stage 3, and the change of the electron diffraction pattern can be easily predicted, and the sample stage is oriented. This is because it is possible to more easily determine the direction in which the camera is located. Considering these, it is desirable that the single crystal portion of the crystalline sample base 5 is a cubic, tetragonal, orthorhombic or hexagonal crystal system. In addition, it is more effective to adopt a configuration that takes into account the cross-sectional shape of the removable support 4 and the crystal system of the single crystal portion.

  FIG. 3 is a schematic view showing the crystal axis direction of the crystalline sample base in the present invention. FIG. 3 is a view of the sample table viewed from the direction of the rotation axis 9 of the sample table as indicated by the arrow in FIG. When the cross-sectional shape of the detachable support 4 is a square, a material whose single crystal portion is any one of cubic, tetragonal and orthorhombic is selected, and the c-axis is directed in the direction of the rotation axis 9 of the sample stage 3. The crystalline sample pedestal 5 is disposed at the end of the detachable support 4 so that the a-axis is in the direction of the normal 10 of the support base (FIG. 3A). Then, in a state where the sample moving mechanism 18 is not inclined, the [100] incident electron diffraction pattern can be used as a reference. Next, when the sample stage 3 is removed, rotated by 90 ° in the direction of the rotation axis, and mounted again (FIG. 3B), the electron diffraction pattern of [010] incidence can be used as a reference. Of course, if the crystalline sample pedestal 5 is arranged at 45 ° with the c-axis in the direction of the rotation axis 9 of the sample table 3 and the a-axis and b-axis in the direction of the support base normal 10, [110] can be used as a reference (FIG. 3C) and [−110] incident electron diffraction patterns can be used as a reference after rotation (FIG. 3D). Alternatively, when the cross-sectional shape of the detachable support 4 is a regular hexagon, a simple electron diffraction pattern can be used as a reference if a material whose single crystal portion is hexagonal is selected. In this way, the sample orientation can be determined using a simpler electron diffraction pattern by successfully combining the cross-sectional shape of the detachable support 4 and the crystal system selection of the single crystal portion of the crystalline sample base 5 and the arrangement in the axial direction. .

  As a material for the crystalline sample base 5, any material of the above-described crystal type can be used. However, considering availability, ease of processing, and stability, metals such as silicon, copper, molybdenum, and gold are used. Is suitable. In particular, silicon is preferable from the viewpoint of availability and workability.

  Since the single crystal portion of the crystalline sample base 5 is used for observing an electron diffraction pattern with a transmission electron microscope, the thickness in the direction of incidence of the electric wire must be such that the electron beam is transmitted. Assuming that it is used in a general transmission electron microscope, it is desirable that the silicon has a shape such that the thickness in the electron beam incident direction is about 300 nm. Therefore, the shape of the crystalline sample base 5 is preferably a prismatic shape or a cylindrical shape with a diameter of about 300 nm. Further, it is desirable that the tip 8 be flat so that the sample 8 can be easily fixed to the tip. Of course, the fixing of the sample 8 is not limited to the tip of the crystalline sample pedestal 5, but since the crystalline sample pedestal 5 and the sample 8 can be close to each other, orientation accuracy and reproducibility can be improved. The tip of the pedestal 5 preferably has a shape that can easily fix the sample 8.

  Such a sample stage 3 can be produced by various methods. For example, a method of fixing a small piece lifted out from a silicon wafer by a focused ion beam processing device to the tip of a copper detachable support 4 processed into a rectangular column shape, and further forming the small piece into a prismatic shape or a cylindrical shape by the focused ion beam processing device. . Alternatively, a method for forming a silicon wafer by forming a projection that becomes a portion of the crystalline sample base 5 in a photolithography process, bonding the silicon wafer and a copper plate, and then cutting the wafer with a dicing saw. Alternatively, by cutting a silicon wafer on which a projection serving as a portion of the crystalline sample pedestal 5 is cut with a dicing saw, the detachable support 4 and the crystalline sample pedestal 5 are formed into a sample table 3 having a shape integrally formed with silicon. There are methods.

  Since such a sample support 1 has a simple configuration and can be reduced in size, it can be mounted on various sample holders. For example, a uniaxial tilt holder or a biaxial tilt holder that can tilt the sample around one axis or two independent axes, a rotating holder that can rotate the sample in a plane that includes the uniaxial tilt and the tilt axis, etc. It is. Among these, the uniaxial tilt holder and the biaxial tilt holder are standard sample holders that are frequently used as standard, and the fact that they can be used with these sample holders increases versatility. By mounting the sample holder 19 that can tilt the sample around at least one axis and combining the rotation mechanism of the sample stage 3 and the tilt mechanism of the sample holder 19, the sample can be observed from all 360 ° directions. It becomes possible. That is, if the sample stage 3 can be rotated every 90 °, the observation with the sample holder 19 tilted in a range of ± 45 ° may be performed twice. If the sample stage 3 can be rotated every 60 °, observation with the sample holder 19 tilted in a range of ± 30 ° may be performed three times. Of course, the sample table 3 can be observed from all directions in the same way even when the rotation step of the sample stage 3 is at other angles.

  Next, the process of the three-dimensional observation method using the present invention will be described with reference to FIG. (1) The sample stage 3 on which the sample 8 is fixed is mounted on the support base 2 (FIG. 4A). (2) The sample holder 19 on which the sample support 1 is mounted is inserted into the transmission electron microscope 11 and the first observation start position and orientation are determined. (3) The sample holder 19 is tilted based on the electron diffraction pattern of one crystal orientation of the crystalline sample base 5. (4) Determine which crystal orientation of the single crystal portion of the crystalline sample base 5 corresponds to the first observation start orientation determined in (2). This is the first sample orientation. (5) Return to the first observation start position / orientation, and use the sample moving mechanism 18 of the transmission electron microscope 11 to capture a first continuous tilt image up to a certain tilt angle. (6) Once the sample holder 19 is removed from the transmission electron microscope 11, the sample support 1 is removed from the sample holder 19. (7) The sample stage 3 is removed from the support base 2, and the sample stage 3 is rotated around the rotation axis 9 of the sample stage 3 (FIG. 4B). Thereafter, the sample stage 3 is mounted on the support base 2 again (FIG. 4C). (8) The sample holder 19 on which the sample support 1 is mounted is inserted into the transmission electron microscope 11 and the second observation start position and orientation are determined. (9) The sample holder 19 is tilted based on the electron diffraction pattern of another crystal orientation of the crystalline sample base 5. (10) Determine which crystal orientation of the crystal of the crystalline sample base 5 is the second observation start orientation determined in (8). This is the second sample orientation. (11) Return to the second observation start position and orientation, and use the sample moving mechanism 18 of the transmission electron microscope 11 to capture a second continuous tilt image up to a certain tilt angle. (12) The tilt angles of the first continuous tilt image and the second continuous tilt image are corrected based on the information on the first sample orientation and the second sample orientation with respect to the single crystal of the crystalline sample base 5. (13) A three-dimensional image is reconstructed from the first continuous tilt image and the second continuous tilt image.

  At this time, one crystal axis of the single crystal portion of the crystalline sample base 5 substantially coincides with the direction of the rotation axis 9 of the sample stage 3, and the other two crystal axes are orthogonal to the direction of the rotation axis 9 of the sample stage. With such a configuration, there is an advantage that the relationship between the sample orientation and the crystal orientation in the step (4) or (10) is simplified, and the correction step in (12) is simplified.

  The sample support according to the present invention can be used for three-dimensional structure observation using a transmission electron microscope because sample observation from 360 ° in all directions can be performed easily and accurately.

DESCRIPTION OF SYMBOLS 1 Sample support body 2 Support base body 3 Sample base 4 Removable support body 5 Crystalline sample base 6 Groove 7 Chord surface 8 Sample 9 Rotating shaft 10 Support base normal 11 Transmission electron microscope 12 Electron gun 13 Converging lens 14 Objective lens 15 Projection lens DESCRIPTION OF SYMBOLS 16 Fluorescent screen 17 Recording apparatus 18 Sample moving mechanism 19 Sample holder 20 Electron beam 21 Sample support mounting part 22 Electron beam optical path 23 Cross-sectional shape of sample stand 24 Cross-sectional shape of groove | channel 25 Space

Claims (5)

  A sample support having a sample stage provided with a crystalline sample pedestal at least a part of which is a single crystal at a tip portion, and a support base to which the sample stage is detachably fitted, the sample stage being rotated A sample support for a transmission electron microscope, characterized in that it can be attached to and detached from a support substrate even if it rotates about an axis.   2. The sample table is detachably fitted in a groove provided in the support base, and the cross-sectional shape of the sample base is the same as the cross-sectional shape of the groove of the support base. A sample support for a transmission electron microscope according to 1.   3. The sample support for a transmission electron microscope according to claim 1, wherein the cross-sectional shape of the sample stage and the cross-sectional shape of the groove of the support base are regular polygons or circles.   4. A transmission electron microscope comprising a sample holder on which a sample is mounted and a sample moving mechanism for adjusting a position and an inclination angle of the sample holder, wherein the sample support is provided on the sample holder. A transmission electron microscope comprising a body.   5. A method for observing a three-dimensional structure of a sample using a transmission electron microscope having a sample holder and a sample moving mechanism according to claim 4, wherein the sample is placed on a sample support provided in the sample holder of the transmission electron microscope. Mounted, and after determining the first sample orientation based on one crystal orientation of the crystalline sample pedestal of the sample support, obtaining a first continuous tilt image group using a sample moving mechanism, Rotate the sample stage of the sample support, determine the second sample direction based on another crystal orientation of the crystalline sample base, and then acquire the second continuous tilt image group using the sample moving mechanism A step of reconstructing a three-dimensional image by identifying the sample orientation of the continuous tilt image by estimating the orientation of each continuous tilt image group from the first sample orientation and the second sample orientation. A method for observing a three-dimensional structure of a sample.

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